1. Field of the Invention
The present invention pertains to a method for treating obesity in a mammal which comprises reducing the biological activity of HMGI genes in the mammal. In another embodiment, the invention pertains to a method for treating a tumor in a patient by reducing the biological activity of normal HMGI genes which comprises administering to the patient a therapeutically effective amount of an inhibitor compound active against normal HMGI-C or HMGI(Y) genes. In another embodiment, the invention pertains to a method of producing a transgenic non-human mammal, the germ cells and somatic cells of which contain an inactivated HMGI gene sequence introduced into the mammal, or an ancestor of the mammal, at an embryonic stage. In another embodiment, the invention pertains to a method for screening candidate compounds capable of inhibiting the biological activity of normal HMGI proteins, or a fragment thereof, which comprises the steps of (a) incubating a HMGI protein, or a fragment thereof, with a candidate compound under conditions which promote optimal interaction; and (b) measuring the binding affinity of the candidate compound to the HMGI protein, or a fragment thereof; and (c) determining from the binding affinity which candidate compounds inhibit the biological activity of HMGI proteins, or a fragment thereof. In another embodiment, the invention pertains to a method for screening candidate compounds capable of inhibiting the biological activity of normal HMGI genes which comprises the steps of (a) transfecting into a cell a DNA construct which contains a reporter gene under control of a normal HMGI protein-regulated promoter; (b) administering to the cell a candidate compound; (c) measuring the levels of reporter gene expression; and (d) determining from the levels of reporter gene expression which candidate compounds inhibit the HMGI biological activity. In another embodiment, the invention pertains to a method for detecting normal HMGI proteins as a diagnostic marker for a tumor using a probe that recognizes normal HMGI proteins, which comprises the steps of (a) contacting normal HMGI proteins from a sample from a patient with a probe which binds to HMGI proteins; and (b) analyzing for normal HMGI proteins by detecting levels of the probe bound to the normal HMGI proteins, wherein the presence of normal HMGI proteins in the sample is positive for a tumor. In another embodiment, the invention pertains to a method for detecting antibodies to normal HMGI proteins using a probe that recognizes antibodies to HMGI normal proteins, which comprises the steps of (a) treating a sample from a patient with a probe which binds to antibodies to normal HMGI proteins; and (b) analyzing for antibodies to HMGI proteins by detecting levels of the probe bound to the antibodies to HMGI proteins, wherein the presence of antibodies to normal HMGI proteins in the sample is positive for a tumor. In another embodiment, the invention pertains to HMGI genes and proteins for use as a starting point to isolate downstream target genes regulated by the HMGI genes and proteins.
2. Description of the Background
The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference and, for convenience, are referenced in the following text and respectively grouped in the appended bibliography.
Understanding various genes and pathways underlying development of multicellular organisms provide insights into the molecular basis of the highly regulated processes of cellular proliferation and differentiation. In turn, genetic aberrations in control of cell growth lead to a variety of developmental abnormalities and, most prominently, cancer (Aaronson, 1991). To pursue identification of genes involved in these fundamental biological processes, the viable pygmy mutation (MacArthur, 1944) was investigated because it gives rise to mice of small stature due to a disruption in overall growth and development of the mouse. An insertional transgenic mutant facilitated cloning of the locus (Xiang et al., 1990) and subsequently it was shown that expression of the HMGI-C gene was abrogated in three pygmy alleles (unpublished results).
HMGI-C belongs to the HMG (high mobility group) family of DNA-binding proteins which are abundant, heterogeneous, non-histone components of chromatin (Grosschedl et al., 1994). HMG proteins are divided into three distinct families, the HMG box-containing HMG1/2, the active chromatin associated HMG14/17 and the HMGI proteins (Grosschedl et al., 1994). At present, the last family consists of two genes, HMGI(Y) (Johnson et al., 1988; Friedmann et al., 1993) which produces two proteins via alternative splicing (Johnson et al., 1989) and HMGI-C (Manfioletti et al., 1991; Patel et al., 1994). A prominent feature of HMGI proteins is the presence of DNA-binding domains which bind to the narrow minor groove of A-T rich DNA (Reeves and Nissen, 1990) and are therefore referred to as A-T hooks. Recently, valuable insights have been gained into their mechanism and role in transcription (Thanos and Maniatis, 1992; Du et al., 1993). The HMGI proteins have no transcriptional activity per se (Wolffe, 1994), but through protein-protein and protein-DNA interactions organize the framework of the nucleoprotein-DNA transcriptional complex. This framework is attained by their ability to change the conformation of DNA and these proteins are therefore termed architectural factors (Wolffe, 1994). In the well-studied case of HMGI(Y) and the interferon B promoter, HMGI(Y) stimulates binding of NF-KB and ATF-2 to appropriate sequences and alters the DNA structure which allows the two factors to interact with each other and presumably with the basal transcription machinery (Thanos and Maniatis, 1992; Du et al., 1993).
A number of studies have revealed an association between increased expression levels of HMGI proteins and transformation (Giancotti et al., 1987, 1989, 1993). For example, in chemically, virally or spontaneously derived tumors, appreciable expression of HMGI-C was found in contrast to no detectable expression in normal tissues or untransformed cells (Giancotti et al., 1989). A recent study has demonstrated a more direct role for HMGI-C in transformation (Berlingieri et al., 1995). Cells infected with oncogenic retroviruses failed to exhibit various phenotypic markers of transformation if HMGI-C protein synthesis was specifically inhibited.
DNA probes adjacent to HMGI-C were mapped to the distal portion of mouse chromosome 10 in a region syntenic to the long arm of human chromosome 12 including and distal to band q13 (Justice et al., 1990). This genomic region is under intensive investigation because it is the location of consistent rearrangements in a number of neoplasms, mainly of mesenchymal origin (Schoenberg Fejzo et al., 1995). Lipomas, tumors mainly composed of mature fat cells, are one of the most common mesenchymal neoplasms that occur in humans (Sreekantaiah et al., 1991). Approximately 50% of lipomas are characterized by cytogenetic rearrangements and the predominant alteration is a presumably balanced translocation involving 12q14-15 with a large variety of chromosomal partners including 1, 2, 3, 4, 5, 6, 7, 10, 11, 13, 15, 17, 21, and X (Sreekantaiah et al., 1991; Fletcher et al., 1993). This variability in reciprocal translocations along with duplications, inversions, and deletions of 12q14-15 in these tumors, strongly indicates a primary role of a gene on chromosome 12 in lipomas. Furthermore, this gene may play a key role in normal differentiation of primitive mesenchyme as not only lipomas, but also uterine leiomyomas (smooth muscle tumors), lipoleiomyomas (smooth muscle and adipose components), and pulmonary chondroid hamartomas (primitive mesenchyme, smooth muscle, adipose, and mature cartilage components) are all clonal proliferations that are characterized by rearrangements of 12q14-15 (Schoenberg Fejzo et al., 1995). Interestingly, breakpoints in a lipoma, a pulmonary chondroid hamartoma and uterine leiomyomata have been shown to map within a single YAC (Schoenberg Fejzo et al., 1995).
The first step in the molecular definition of the pygmy mutation was made possible by the isolation of a transgenic insertional mouse mutant at the locus, pgTgN40ACha (Xiang et al., 1990). A 0.5 kb ApaI-ApaI single copy genomic sequence 2 kb from the site of transgene insertion was identified (Xiang et al., 1990) and used to initiate a bi-directional chromosome walk on normal mouse genomic DNA. The analysis of seven overlapping clones spanning 91 kb delineated a 56 kb common deletion between two informative mutants, pg and pgTgN40ACha (FIG. 8a).
The common area of disruption was investigated further for candidate transcription units. The technique of exon amplification (Buckler et al., 1991) was employed to identify putative exons and clones 803 and 5B, in the same orientation, produced spliced products (FIG. 8b). Their sequence was determined (Ausubel et al., 1988) and a comparison to DNA sequence databases (GenBank and EMBL) revealed 100% homology to a previously identified gene, HMGI-C (Manfioletti et al., 1991) (FIG. 8c). The HMGI members have been assigned multiple functions (Manfioletti et al., 1991) and recently, have been shown to play a critical role in regulation of gene expression as architectural factors by inducing DNA conformational changes in the formation of the three-dimensional transcription complex (Thanos and Maniatis, 1992; Du, W. et al., 1993).
Subsequently, the genomic structure of HMGI-C revealed that the gene contains five exons and spans a region of approximately 110 kb (FIG. 8d). Single copy sequences from the 190 kb cloned pygmy locus, surrounding and including the HMGI-C gene (FIG. 8d), were used as probes on Southern blots containing DNA isolated from the two informative alleles (Xiang et al., 1990). The genomic area encompassing HMGI-C is completely deleted in the transgenic insertional mutant pgTgN40ACha (A/A), whereas in the spontaneous mutant pg, the 5xe2x80x2 sequences and the first two exons are absent (FIG. 8d).
Cancer arises from aberrations in the genetic mechanisms that control growth and differentiation and ongoing elucidation of these mechanisms continues to improve the understanding of mammalian development and its various abnormalities. Increasingly, accumulating experimental evidence points towards transcriptional deregulation as one of the pivotal events in neoplasia. Many of the known transforming retroviral oncogenes, such as v-myc, v-fos and v-myb, are homologs of mammalian transcription factors which are normally involved in proliferation and differentiation control. Genes that encode for such transcription factors are frequently affected by the somatically acquired genetic changes which arise stochastically over a lifetime of an organism. These alterations, which can either activate expression of the relevant genes or disrupt them to create novel fusion proteins, affect transcription networks and initiate cancer.
One of the transcription factors whose disruption was shown to result in tumorigenesis is HMGI-C, which has attracted considerable attention for two reasons. First, a series of elegant experiments demonstrated that HMGI(Y) is involved in transcriptional regulation and is required for virus induction of the human interferon-xcex2 gene expression. These obserations were incorporated into a novel model in which activation of gene expression is initiated by a higher order transcription enhancer complex. This functional nucleoprotein entity termed enhanceosome is formed when several distinct transcription factors assemble on DNA in a stereospecific manner. Combinatorial mechanisms of the enhanceosome formation enable the cell to achieve high specificity of gene activation in response to multiple biological stimuli. As an essential component of the enhanceosome, HMGI(Y) promotes the assembly of this three-dimensional structure through both protein-protein and protein-DNA interactions. The latter activity is mediated through the HMGI DNA-binding domains.
The function of HMGI-C, the other known member of the HMGI family, in growth and development control is better understood at the biological level. In humans, rearrangements of HMGI-C were linked to the pathogenesis of several distinct types of solid tumors. Rearrangements of the chromosomal band 12q13-15, consistently found in a wide variety of benign mesenchymal neoplasms, disrupt HMGI-C and generate novel chimeric transcripts. In the vast majority of the analyzed tumors, these transcripts consist of the HMGI-C DNA-binding domains fused to ectopic sequences provided by the translocation partner.
In the mouse, HMGI-C inactivation produced a dramatic disruption of both pre- and postnatal growth, resulting in the pygmy phenotype. Pygmy mice exhibit significant growth retardation which is first apparent in midgestation and becomes even more pronounced after birth. Adult animals are proportionally built and viable but exhibit a 60% weight reduction compared to their wildtype littermates. A detailed phenotypic analysis of the pygmy mouse revealed that the weight reduction in most of the tissues is commensurate with the overall decrease in body weight. Most interestingly, HMGI-C inactivation does not affect the growth hormone-insuline-like growth factor endocrine pathway, suggesting that HMGI-C functions in a previously unknown growth regulatory mechanism.
The molecular basis of the pygmy mutation is not well understood. High levels of the HMGI proteins are not required for cell growth per se and elevated HMGI expression appear to be associated with the biological state of the cell more directly than with its high proliferation rate. Upon transformation with oncogenic retroviruses, expression of HMGI-C and HMGI(Y) in epithelial cells is dramatically increased even though the proliferative capacity of the infected cells remains unaffected. Furthermore, analysis of a transformed cell line which retained its differentiated phenotype revealed that levels of the HMGI expression were significantly lower than in cell lines which lost their differentiation markers as a result of transformation. Other studies demonstrated that HMGI-C is expressed in less differentiated mesenchymal cells but is no longer present in their terminally differentiated counterparts. In combination, these results indicate that the function of the HMGI proteins may be to maintain the undifferentiated cellular state.
The diverse set of mesenchymal neoplasms in which HMGI-C is frequently disrupted by translocations of 12q13-15 includes lipomas, uterine leiomyoma, pulmonary hamartoma and pleomorphic adenomas of salivary gland. Another cytogenetic subgroup which can be identified in this set of tumors is characterized by rearrangements at 6p21-23. Intriguingly, HMGI(Y) has previously been localized to this chromosomal area.
Uterine leiomyomata, also known as fibroids, are the most common pelvic tumors in women. Systematic histologic examination of hysterectomy specimens has shown a prevalence as high as 77% for these tumors in women of reproductive age. Although benign, uterine leiomyomata constitute a major health problem as they are associated with abnormal uterine bleeding, pelvic pain, urinary incontinence, spontaneous abortion, premature delivery, and infertility. Symptomatic fibroids are the leading indication for hysterectomy, accounting for 27% of the estimated 680,000 procedures performed annually in the United States.
Several different consistent chromosomal rearrangements have been identified in uterine leiomyomata, and they suggest involvement of a critical gene on chromosome 12 in the pathobiology. A translocation involving chromosomes 12 and 14, t(12;14)(q14-15;q23-24), represents one of the most common rearrangements, although trisomy 12, inversions and duplications of 12q14-q15, and translocations of 12q14-q15 with chromosomes other than 14 are not uncommon. The breakpoint in 12q14-q15 in uterine leiomyomata is in an intriguing chromosomal region because it is also the location of consistent rearrangements in other benign solid tumors, including lipomas and pleomorphic adenomas of the salivary gland. Rearrangements of 12q13-15 have been reported in pulmonary chondroid hamartoma, endometrial polyps, epithelial breast tumors, hemangiopericytoma, and an aggressive angiomyxoma. These tumors have the common properties of being mesenchyme-derived and benign. Therefore, it has been hypothesized that a single gene involved in mesenchyme differentiation and growth could be responsible for these multiple tumor types.
H. R. Asher et al. (1995) reported that HMGI-C, an architectural factor that functions in transcriptional regulation, is disrupted by rearrangement at the 12q14-15 chromosomal breakpoint in lipomas and suggests a role for HMGI-C in adipogenesis and mesenchyme differentiation.
X. Zhou et al., (1995) shows that the pygmy phenotype arises from the inactivation of HMGI-C which function as architectural factors in the nuclear scaffold and are critical in the assembly of stereospecific transcriptional complexes.
A. C. Finlay et al. (1951) discloses an antibiotic obtained from culture filtrates of an Actinomycete, Streptomyces netropsis, isolated from a soil sample and assigned the name Netropsin.
A. DiMarco et al. (1962) disclose the physicochemical properties of, and the results obtained against some experimental tumors with, the antibiotic of the netropsin group, distamycin A. Distamycin A is reported to exhibit a strong inhibition on ascites tumors [Ehrlich and sarcoma 180 (S180)] and delays the growth of solid tumors (Ehrlich carcinoma, S180, Walker carcinoma, and Oberling-Guerin-Guerin myeloma). Distamycin A is also reported to decrease the mitotic index of the Ehrlich ascites tumor and induces mitotic damages of tumor cells.
M. L. Kopka et al. (1985) discloses that X-ray analysis of the complex of netropsin with the B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-BrC-G-C-G (SEQ ID NO:33) reveals that the antitumor antibiotic binds within the minor groove by displacing the water molecules of the spine of hydration. Netropsin amide NH is reported to furnish hydrogen bonds to bridge DNA adenine N-3 and thymine O-2 atoms occurring on adjacent base pairs and opposite helix strands, exactly as with the spine of hydration.
R. Reeves et al. (1990) discloses the domains of the mammalian high mobility group (HMG)I chromosomal proteins necessary and sufficient for binding to the narrow minor groove of stretches of A T-rich DNA. The three highly conserved regions within each of the known HMG-I proteins is reported to be closely related to the consensus sequence T-P-K-R-P-R-C-R-P-K-K (SEQ ID NO:34) and that a synthetic oligopeptide corresponding to this consensus xe2x80x9cbinding domainxe2x80x9d (BD) sequence specifically binds to substrate DNA in a manner similar to the intact HMG-I proteins. Molecular Corey-Pauling-Koltun model building and computer simulations employing energy minimization programs to predict structure are reported to suggest that the consensus BD peptide has a secondary structure similar to the antitumor and antiviral drugs netropsin and distamycin, and to the dye Hoechst 33258 and that in vitro these ligands, which are preferentially bind to A T-rich DNA, have been demonstrated to effectively compete with both the BD peptide and the HMG-I proteins for DNA binding. The BD peptide is also reported to contain novel structural features such as a Asx bend of xe2x80x9chookxe2x80x9d at its amino-terminal end and laterally projecting cationic Arg/Lys side chains or xe2x80x9cbristlesxe2x80x9d which may contribute to the binding properties of the HMGI proteins. The predicted BD peptide structure, referred to as the xe2x80x9cA T-hook,xe2x80x9d represents a DNA-binding motif capable of binding to the minor groove of stretches of A T base pairs.
European patent EP727487A1 (960821) (Bullerdiek et al.) discloses the Multi-tumor Aberrant Growth (MAG) gene having the nucleotide sequence of any one of the strands of any one of the members of the High Mobility Group protein genes or LIM protein genes.
The present invention pertains to a method for treating obesity in a mammal which comprises reducing the biological activity of HMGI genes in the mammal. In this embodiment, at least 10% of the biological activity of HMGI genes is reduced, and preferably at least 50% of the biological activity of HMGI genes is reduced. In one embodiment, the biological activity of HMGI-C genes is reduced, and in another embodiment, the biological activity of HMGI-(Y) genes is reduced. The mammal is preferably leptin-deficient or leptin receptor-deficient. The reduction in biological activity of HMGI genes may be achieved by inhibiting the expression of HMGI genes, by administering to the mammal a therapeutically effective amount of an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA of the HMGI gene, by inhibiting the DNA-binding activity of HMGI genes, by administering to the mammal a therapeutically effective amount of an inhibitor compound selected from the group consisting of netropsin, distamycin A, or Hoechst 33258 (bisbenzimide), or by inhibiting the protein-protein interactions of HMGI proteins. The mammal may be a human or a rodent. The biological activity of HMGI genes may also be substantially reduced by breeding the mammal with an inactivated HMGI gene sequence introduced into the mammal, or an ancestor of the mammal, at an embryonic stage. The inactivated HMGI gene sequence may be an inactivated HMGI-C gene sequence and may be the inactivated HMGI-C gene sequence set out in FIG. 10.
In another embodiment, the present invention pertains to a method for treating a tumor in a patient by reducing the biological activity of normal HMGI genes which comprises administering to the patient a therapeutically effective amount of an inhibitor compound active against normal HMGI-C or HMGI(Y) genes. In this embodiment, the biological activity of normal HMGI-C genes may be reduced or the biological activity of normal HMGI-(Y) genes may be reduced. The reduction in biological activity of normal HMGI genes may be achieved by inhibiting the expression of normal HMGI genes, by administering to the patient a therapeutically effective amount of an oligonucleotide which has a nucleotide sequence complementary to at least a portion of the mRNA of the normal HMGI gene, by inhibiting the DNA-binding activity of normal HMGI genes, or by administering to the patient a therapeutically effective amount of an inhibitor compound selected from the group consisting of netropsin, distamycin A, or Hoechst 33258 (bisbenzimide). In one embodiment, the tumor is mesenchyme-derived and benign and may be uterine leiomyomata, lipomas, pleomorphic adenomas of the salivary gland, pulmonary chondroid hamartoma, endometrial polyps, epithelial breast tumors, hemangiopericytoma, or angiomyxoma, and is preferably uterine leiomyomata, lipomas, or pleomorphic adenomas of the salivary gland. In another embodiment, the tumor is a malignant tumor of epithelial origin and may be a carcinoma of the lung, colon, breast, prostate, thyroid gland, or skin. The reduction in biological activity of normal HMGI genes may be achieved by inhibiting the protein-protein interactions of HMGI proteins.
In yet another embodiment, the present invention pertains to a method of producing a transgenic non-human mammal, the germ cells and somatic cells of which contain an inactivated HMGI gene sequence introduced into the mammal, or an ancestor of the mammal, at an embryonic stage. In this embodiment, the inactivated HMGI gene sequence may be an inactivated HMGI-C gene sequence and may be the inactivated HMGI-C gene sequence set out in FIG. 10. Preferably, the genome of the mammal does not encode for both the functionally active leptin gene and the functionally active HMGI genes.
In yet another embodiment, the present invention pertains to a method for screening candidate compounds capable of inhibiting the biological activity of normal HMGI proteins, or a fragment thereof. The method comprises the steps of (a) incubating a HMGI protein, or a fragment thereof, with a candidate compound under conditions which promote optimal interaction; and (b) measuring the binding affinity of the candidate compound to the HMGI protein, or a fragment thereof; and (c) determining from the binding affinity which candidate compounds inhibit the biological activity of HMGI proteins, or a fragment thereof. The candidate compound may inhibit the biological activity of normal HMGI proteins, or a fragment thereof, in an amount of at least 10%. The binding affinity may be measured using a scintillation proximity assay or a fluorescence polarization assay.
In yet another embodiment, the present invention pertains to a method for screening candidate compounds capable of inhibiting the biological activity of normal HMGI genes. The method comprises the steps of (a) transfecting into a cell a DNA construct which contains a reporter gene under control of a normal HMGI protein-regulated promoter; (b) administering to the cell a candidate compound; (c) measuring the levels of reporter gene expression; and (d) determining from the levels of reporter gene expression which candidate compounds inhibit the HMGI biological activity. In this embodiment, the candidate compound may inhibit the biological activity of normal HMGI genes in an amount of at least 10%.
In yet another embodiment, the present invention pertains to a method for detecting normal HMGI proteins as a diagnostic marker for a tumor using a probe that recognizes normal HMGI proteins. The method comprises the steps of (a) contacting normal HMGI proteins from a sample from a patient with a probe which binds to HMGI proteins; and (b) analyzing for normal HMGI proteins by detecting levels of the probe bound to the normal HMGI proteins, wherein the presence of normal HMGI proteins in the sample is positive for a tumor. In this embodiment, normal HMGI-C proteins may be detected or normal HMGI(Y) proteins may be detected. In one embodiment, the tumor is mesenchyme-derived and benign and may be uterine leiomyomata, lipomas, pleomorphic adenomas of the salivary gland, pulmonary chondroid hamartoma, endometrial polyps, epithelial breast tumors, hemangiopericytoma, or angiomyxoma. In another embodiment, the tumor is a malignant tumor of epithelial origin and may be a carcinoma of the lung, colon, breast, prostate, thyroid gland, or skin. The probe may be an antibody, the sample may be a biopsy sample, a urine sample, a blood sample, a feces sample, or a saliva sample, and the method may be a histological assay, biochemical assay, flow cytometry assay, Western blot assay, or solution assay. A positive and negative control sample may be treated according to the method to assess the level of normal HMGI proteins in a tumor sample and a nontumor sample, respectively.
In yet another embodiment, the present invention pertains to a method for detecting antibodies to normal HMGI proteins using a probe that recognizes antibodies to HMGI normal proteins. The method comprises the steps of (a) treating a sample from a patient with a probe which binds to antibodies to normal HMGI proteins; and (b) analyzing for antibodies to HMGI proteins by detecting levels of the probe bound to the antibodies to HMGI proteins, wherein the presence of antibodies to normal HMGI proteins in the sample is positive for a tumor. In this embodiment, antibodies to normal HMGI-C may be detected or antibodies to normal HMGI(Y) may be detected. The probe may be normal HMGI-C or HMGI(Y) proteins. In one embodiment, the tumor is mesenchyme-derived and benign and may be uterine leiomyomata, lipomas, pleomorphic adenomas of the salivary gland, pulmonary chondroid hamartoma, endometrial polyps, epithelial breast tumors, hemangiopericytoma, or angiomyxoma. In another embodiment, the tumor is a malignant tumor of epithelial origin and may be a carcinoma of the lung, colon, breast, prostate, thyroid gland, or skin. The sample may be a biopsy sample, a urine sample, a blood sample, a feces sample, or a saliva sample and the method may be a histological assay, biochemical assay, flow cytometry assay, Western blot assay, or solution assay.
In yet another embodiment, the present invention pertains to HMGI genes and proteins for use as a starting point to isolate downstream target genes regulated by the HMGI genes and proteins.